Abstract With the information we currently have available, a vaccine that prevents HIV infection outright in a substantial portion of vaccinated individuals seems out of reach. A far more realistic vaccine would be one that blunts acute viremia and, more importantly, maintains immunologic suppression of the virus for long periods of time without the aid of antiretroviral drugs. Such a vaccine should elicit potent T cell responses. The CMV vectored SIV vaccine has reinvigorated hopes that such a vaccine could be possible. One key to the success of the CMV vectored vaccine is its remarkable ability to prime and maintain CD8 T cell responses against SIV that are restricted in non-canonical fashion, specifically targeting SIV derived epitopes that are presented in the context of MHC-II and MHC-E molecules. However, it?s not clear at this time that the CMV vectored vaccine can be directly translated to HIV. Thus, more vaccine candidates are sorely needed that induce similar responses so that basic mechanistic experiments can be conducted to understand how other vaccine platforms can be made to induce similarly effective CD8 T cell responses. To this end, we have investigated a novel attenuated mutant of SIV that lacks just two amino acids in the Env cytoplasmic domain. This virus, termed ?GY, induces potent T cell responses along with no neutralizing antibodies, and protects pigtail macaques from multiple homologous and heterologous pathogenic SIVs. Remarkably, our preliminary data suggest ?GY, like CMV, elicits non-canonical CD8 T cell responses that, in vitro, can target and eliminate virus infected target cells. Here, we propose to capitalize on this phenomenon to begin to tease out these responses. To do this, we will use Mauritian cynomolgus macaques (MCM), which, in stark contrast to pigtails, exhibit remarkably simple MHC genetics due to a population bottleneck on the island. In Aim 1, we will vaccinate MCM with ?GY and comprehensively characterize the CD8 T cell response with focus on alternately restricted T cells, including determining their molecular restriction. In Aim 2, we will assess whether these responses can protect this species of macaque from pathogenic SIVsmE660. We will also expand T cell lines and clones in vitro from the vaccinated animals and determine their capacity to eliminate diverse pathogenic challenge viruses in vitro. Together, this highly focused experimental design will allow us to begin to dissect non-canonical CD8 T cells, their restricting molecules, and their ability to protect from diverse viruses. We hope these data will lead to larger projects designed to decipher the mechanisms, perhaps shared with CMV, that lead to these highly potent alternate CD8 T cell responses.